Acetylation of Cyclic AMP Receptor Protein by Acetyl Phosphate Modulates Mycobacterial Virulence

Abstract

The success of Mycobacterium tuberculosis (Mtb) as a pathogen is partly attributed to its ability to sense and respond to dynamic host microenvironments. The cyclic AMP (cAMP) receptor protein (CRP) is closely related to the pathogenicity of Mtb and plays an important role in this process. However, the molecular mechanisms guiding the autoregulation and downstream target genes of CRP while Mtb responds to its environment are not fully understood. Here, it is demonstrated that the acetylation of conserved lysine 193 (K193) within the C-terminal DNA-binding domain of CRP reduces its DNA-binding ability and inhibits transcriptional activity. The reversible acetylation status of CRP K193 was shown to significantly affect mycobacterial growth phenotype, alter the stress response, and regulate the expression of biologically relevant genes using a CRP K193 site-specific mutation. Notably, the acetylation level of K193 decreases under CRP-activating conditions, including the presence of cAMP, low pH, high temperature, and oxidative stress, suggesting that microenvironmental signals can directly regulate CRP K193 acetylation. Both cell- and murine-based infection assays confirmed that CRP K193 is critical to the regulation of Mtb virulence. Furthermore, the acetylation of CRP K193 was shown to be dependent on the intracellular metabolic intermediate acetyl phosphate (AcP), and deacetylation was mediated by NAD⁺-dependent deacetylases. These findings indicate that AcP-mediated acetylation of CRP K193 decreases CRP activity and negatively regulates the pathogenicity of Mtb. We believe that the underlying mechanisms of cross talk between transcription, posttranslational modifications, and metabolites are a common regulatory mechanism for pathogenic bacteria. IMPORTANCE Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, and the ability of Mtb to survive harsh host conditions has been the subject of intensive research. As a result, we explored the molecular mechanisms guiding downstream target genes of CRP when Mtb responds to its environment. Our study makes a contribution to the literature because we describe the role of acetylated K193 in regulating its binding affinity to target DNA and influencing the virulence of mycobacteria. We discovered that mycobacteria can regulate their pathogenicity through the reversible acetylation of CRP K193 and that this reversible acetylation is mediated by AcP and a NAD⁺-dependent deacetylase. The regulation of CRP_Mtb by posttranslational modifications, at the transcriptional level, and by metabolic intermediates contribute to a better understanding of its role in the survival and pathogenicity of mycobacteria.

Keywords: CRP; Mycobacterium tuberculosis; cAMP; cAMP receptor protein; cyclic AMP; lysine acetylation; virulence.

FIG 1

Acetylation of K193 site affects the DNA-binding ability of CRP. (a) DNA-binding abilities of CRP^WT and CRP mutants. EMSA was used to evaluate the DNA-binding abilities of CRP^WT and its derivatives at the indicated concentrations (0, 5, and 15 μM) to biotin-labeled target DNA 2D3 following a 20-min incubation. Lane 1 represents the labeled DNA alone. (b) DNA-binding abilities of CRP^WT were evaluated with different concentrations of cAMP. (c) Effect of cAMP on DNA-binding abilities of CRP^K193R, CRP^K193Q, and CRP^K193A evaluated by EMSA. (d) Western blot analysis of purified CRP^WT and CRP^K193Ace using a specific antibody against the acetylated K193 residue of CRP_Mtb, in which K193 was successfully acetylated (CRP^K193Ace). Antibodies specific for CRP (anti-CRP; 1:2,000) and acetylated K193 peptides (anti-K193^Ace; 1:2,000) were used. (e) DNA-binding abilities of CRP^K193Ace and CRP^WT. The binding of CRP^WT to the target DNA 2D3 could be completed by a 500-fold excess of unlabeled 2D3 DNA as shown in lane 5 of panel a and lane 8 of panel e. The concentrations of cAMP and proteins used in the EMSA are indicated, and each EMSA result is representative of three independent replicates. K193Q, substitution of K193 with glutamine to mimic acetylation; K193R, substitution of K193 with arginine to mimic deacetylation; K193A, substitution of K193 with glycine.

FIG 2

Positive charge of CRP K193 is essential for its transcriptional activity. The transcription levels of CRP_Msm (MSMEG_6189) and its target genes in chromosome CRP variants. MSMEG_3197 and MSMEG_6190 in Msm correspond to Rv1592c and Rv3677 in Mtb, respectively. The transcript levels were determined by qPCR and 2^−ΔΔCT methods. Each experiment consisted of three independent replicates. The expression of the tested gene was normalized to that of 16S rRNA and compared to that in Msm; significance was determined by ANOVA. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 3

Growth phenotypes of CRP K193 site-specific mutation strains. (a) Growth curves of Msm-CRP^WT, Msm-CRP^K193R, and Msm-CRP^K193Q in 7H9-OADC medium. Data are means and standard deviations (SD) (n ≥ 3). (b and c) Colony morphology (b) and biofilm formation (c) of Msm-CRP^WT, Msm-CRP^K193R, and Msm-CRP^K193Q. The colonies were grown in 7H10-OADC medium for 4 days at 37°C. Biofilms were grown at the air-medium interface in 12-well tissue culture plates in Sauton’s medium for 4 days at 37°C. (d) Morphological observations of Msm. (e) Lengths of Msm bacterial cells. Each experiment consisted of three independent replicates. K193R, substitution of K193 with arginine to mimic deacetylation; K193Q, substitution of K193 with glutamine to mimic acetylation.

FIG 4

Acetylation of CRP K193 regulates the resistance of mycobacteria to external stress environments. (a to f) Survival ability of mutant strains exposed to different stress conditions, including 0.05% SDS (a), 5 mM H₂O₂ (b), hypoxia (c), low pHs of 3.5 (d) and 4.5 (e), and heat treatment (f). After treatment, bacteria were serially diluted 10-fold and plated on 7H10-OADC for determination of the number of CFU per milliliter. Error bars represent SD from at least three independent experiments. (g) Expression levels of CRP and acetylation levels of K193 under different conditions. The loading control for panels g and h is shown in Fig. S3a and b. (h) Expression of CRP and acetylation levels of K193 with different concentrations of cAMP. CRPs were immunoprecipitated using the anti-CRP antibody (1:2,000) and the specific anti-K193^Ace (1:2,000) acetylation antibody. Relative intensity represents the ratio of grayscale between different stress conditions and the normal condition (7H9-OADC). Relative K193^Ace-CRP represents the grayscale ratio of K193 acetylation level to CRP level. Western blots were repeated independently at least three times. ns, not significant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

FIG 5

Acetylation of CRP K193 modulates bacterial virulence in mouse model. (a) Replication of bacteria in RAW264.7 cells. The net growth between 2 and 24 h was calculated from the fold change in CFU per milliliter recovered at these time points. Error bars represented SD from at least three independent experiments. (b to f) Bacterial burdens in different organs at different time points. The lungs, spleen, and liver were harvested at different time points (8 h, 2 days, 5 days, 8 days, and 11 days) after intravenous injection, and CFU were counted to quantify the number of bacteria in these organs. (g) Survival rates of mice infected by intraperitoneal injection. Eight-week-old C57BL/6 female mice were intravenously administered 1.0 × 10⁸ bacteria in 200 μL PBS. Control mice were given 200 μL PBS. The number of live mice was determined daily. (h) Hematoxylin-and-eosin-stained lungs of bacterium-treated C57BL/6 mice. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 6

Acetylation of CRP K193 is regulated by AcP. (a) Western blotting was used to verify whether CRP can be acetylated by AcP in vitro with the indicated concentrations of AcP (lanes 2, 3, 5, and 6). Lane 1 represents CRP alone. CRP was immunoprecipitated using an anti-His antibody (1:5000). CRP acetylation levels were determined using the pan anti-acetyllysine antibody (1:2,000). (b to e) Intracellular concentrations of AcP at different phases of growth. Mma (b to d) and Mtb (e) cells were harvested at the indicated time points. The intracellular concentrations of AcP at early exponential phase (b), late exponential phase (c), and stationary phase (d) were measured. Error bars indicate SD from triplicate measurements. (f and g) Expression of CRP and the acetylation level of K193 in the wild-type strain and the ackA knockout strain with different carbon sources. Mma (f) and Mtb (g) were cultured in 7H9-AC (BSA and catalase) with different carbon source supplementation. CRP was immunoprecipitated using an anti-CRP antibody (1:2,000), and K193 acetylation level was determined using the anti-K193^Ace antibody (1:2,000). Relative intensity represents the ratio of grayscale between the ackA knockout strain and the WT with each carbon source. Relative K193^Ace-CRP represents the grayscale ratio of K193 acetylation to CRP levels. Each experiment consisted of three independent replicates. Statistical significance was determined with Student’s t test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 7

CRP K193 can be deacetylated in vitro and in vivo. (a) Rv1151c deacetylates CRP in a dose-dependent manner in vitro. Purified CRP^K193Ace (5 μM) was incubated with NAD⁺ (1 mM) and Rv1151c (8 μM) at 25°C for 2 h. The acetylation levels were evaluated by Western blotting with anti-CRP (1:2,000) and anti-K193^Ace (1:2,000) antibodies. (b) Acetylation levels of CRP in wild-type H37Ra (Ra) and a MRA_1161 (Rv1151c, homolog in H37Ra) deletion mutant (knockout [KO]) in vivo. (c and d) Expression of Rv1151c under different cAMP concentrations (c) and different stress conditions (d). Cell extracts (20 μg per lane) were analyzed by Western blotting with anti-CRP (1:2,000), anti-K193^Ace (1:2,000), and anti-Rv1151c (1:2,000) antibodies. The loading controls for panels c and d can be found in Fig. S3c and d. Relative intensity represents the ratio of grayscale between different stress conditions and the normal condition (7H9-OADC). Results are representative of three independent experiments.